腺相关病毒标记神经细胞的三光子显微成像

深圳大学物理与光电工程学院,教育部光电子器件与系统/广东省重点实验室,广东深圳518060

光学工程; 三光子显微成像; 脑部注射; 腺相关病毒; 海马体神经细胞; 急性脑切片; 三光子荧光; 三次谐波产生

Three-photon microscopy imaging of adeno-associated viral labeled neurons
TONG Shen, LIU Hongji, and WANG Ke

College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen University, Shenzhen 518060, Guangdong Province, P.R.China

optical engineering; three-photon microscopy imaging; brain injection; adeno-associated virus; hippocampus neurons; acute brain slice; three-photon fluorescence; third harmonic generation

DOI: 10.3724/SP.J.1249.2020.01039

备注

1 700 nm波段三光子荧光成像已成为研究神经细胞的重要手段,但该技术仅能在转基因小鼠中对荧光蛋白标记的神经细胞成像,无法对普通小鼠的脑部神经细胞成像. 结合1 700 nm波段三光子显微成像技术和脑部注射神经细胞标记技术,对普通小鼠急性脑切片中的神经细胞进行三光子荧光成像. 结果表明,脑部注射腺相关病毒(AAV9-hSyn-NES-jRGECO1a-WPRE)能够标记普通小鼠灰质和海马体中的神经细胞. 三光子荧光成像再现了普通小鼠脑片中灰质和海马体神经细胞形态. 同时采集的三次谐波图像实现了普通小鼠脑片内白质等结构的无标记成像,且神经细胞位置处的三光子荧光和三次谐波图像呈互补关系. 1 700 nm波段的三光子显微成像技术,结合脑部注射腺相关病毒神经细胞标记技术,可为神经脑科学研究提供一种新颖的成像手段.

Recently, it has been demonstrated that three-photon fluorescence microscopy excited at 1 700 nm window provides a novel technique for imaging neurons. However, this technique can only be used for genetically modified mice rather than ordinary mice, because neurons from genetically modified mice express fluorescent proteins. In this paper, combining brain injection and three-photon fluorescence microscopy, we image neurons in acute brain slices of ordinary mice. The experimental results show that injection of adeno-associated virus(AAV9-hSyn-NES-jRGECO1a-WPRE)can be used to label neurons in the brain of normal mice. Three-photon fluorescence microcopy can reconstruct the neurons morphology of both the grey matter and hippocampus in common mouse brain slices. The simultaneously acquired label-free third-harmonic image shows the white matter, and is complementary to three-photon fluorescence image in neurons. Three-photon microimaging at 1 700 nm, combined with brain injection of adeno-associated virus, provides a new helpful method for neural brain research.

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